PURPOSE: Tumour metastasis is the most clinically significant and enigmatic aspect of tumour behavior and is an unequivocal hallmark of malignancy. Until recent years little has been known about the transportation phase of vascular dissemination during biopsy, because of the technical difficulties in demonstrating circulating cancer cells.
AIMS: This study examined whether cancer cell dissemination results from incisional biopsy in the peripheral blood by using Cytokeratin 19(CK-19) as the marker for Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). In-house recipes without utilizing kits were employed to extract genomic and total RNA to make the procedure user friendly.
MATERIALS AND METHODS: The study population consisted of n=10patients who were clinically diagnosed for oral squamous cell carcinoma and who had not undergone any previous biopsies. 5 patients who were to undergo incisional biopsies for benign conditions served as controls.5 ml of blood aspirates were collected before and within 15 minutes after incisional biopsy. CK-19 gene and a positive control gene β actin were isolated to confirm the primers. Using the total RNA, RT-PCR was performed for β actin and Ck 19 gene expression.
RESULTS: Rt-PCR did show any expression for the CK-19 gene.
CONCLUSION: In conclusion there was no evidence of dissemination of cancer cells in our study and the patients are on a regular follow up for the past one and half years. But larger sample size should be examined to make the procedure a diagnostic tool for cancer metastasis

Keywords: Cytokeratin19, β actin, Rt-pcr

INTRODUCTION

Malignant neoplasms of the oral cavity are relatively rare in most parts
of the world. In countries such as United Kingdom and the United States
for example, malignant neoplasms comprise only 2% to 3% of
total malignant tumours.[1] There
are exceptions however, and in various parts of India and with regard
to Chennai, approximately 40% of all malignant neoplasms are oral in males and around 9% in
females.[2] The vast majority
are epidermoid (squamous cell) carcinomas, accounting for 94.5% of
all cancers of oral cavity.

The size of the clinical problem of oral
squamous cell carcinoma is illustrated in [Table
- 1],
which shows the recent annual average number of new cancer registrations
in our dental college & hospital only.

The role of histopathologist in the evaluation of tumour specimens
has become complex in the last 10-20years and will continue to develop
in
the decade ahead. This is in part is attributable to the increasing
sophistication of therapeutic approaches employed by oncologists, leading
to requirements
for more accurate identification of tumour groups that may vary in
their natural progression and responsiveness to treatment

Tumour metastasis is the clinically significant and enigmatic aspect
of tumour behavior, which can be defined as relentless progress of
neoplasia resulting in death.[3] It
is believed that a few malignant cells are released into the blood
stream during incisional biopsy of malignant epithelial tissues of
the oral
cavity and these free floating cells may establish metastasis elsewhere.[4] Several
authors have tried to show the dissemination phase of cancer cells
in relation to cancers arising from other body tissues.[5], [6], [7] With
the advent of molecular biology several studies have been successfully
tested in detecting micrometastasis especially in relation to lung,
breast and prostrate carcinomas.[8], [9], [10]

Cytokeratins are one of the main families of intermediate filaments
that belong to a multigene family distinguished by their cell type
specific
expression. Malignant cells general retain their intermediate filaments
of their progenitor cell type and consequently cytokeratins have
been used to characterize neoplastic cells.[11], [12] Among
these, Cytokeratin 19, a 40-kDa epithelial cytoskeletol protein is
considered as an epithelial marker as they are not expressed in normal
haematopoietic
tissue.[13], [14]

As very few studies have been carried out in relation to oral cancer
dissemination phase especially in Indian scenario, our objective
was to test if we can detect these released cells by a powerful and
sensitive
method like RT-PCR, an adaptation of Polymerase Chain Reaction (PCR.)

MATERIALS AND METHODS

The study group consisted of n = 10 patients who were clinically diagnosed
for oral cancer and were to undergo incisional biopsy [Table
- 2]. The control group comprised of patients who were to undergo
incisional biopsies for benign conditions (n = 5) [Table
- 3].
The patients in the study group were clinically staged and an informed
consent was obtained from all patients. Subsequently, a 17-gauge needle
was placed in the median forearm vein. To avoid dermal contamination,
initial 1 to 2 ml of blood was discarded and then subsequently 5ml of
blood was collected. Samples of peripheral blood (5ml) were collected
at various times viz. immediately before incision and within 15 minutes
after biopsy. Subsequently 300 aliquots of whole blood was taken in two
microfuge tubes for isolation of genomic DNA along with 900ml of red
blood cell lysis buffer, incubated, centrifuged in microfuge and pellets
of WBC were resuspended and then combined in a single tube. Then 600ml
of ice-cold cell lysis buffer was added, homogenized with subsequent
addition of Dnase-free Rnase and incubated at 37°c for 60 minutes. After adding 200ml of potassium acetate, the solution was vortexed and centrifuged at maximum speed (15,000 rpm) to obtain a visible pellet of the complex. The pellet was then dissolved in isopropanol and then reprecipitated with 70% ethanol. After centrifugation the DNA pellet was air dried and then finally redissolved in 100ml Tris EDTA (pH 7.6). After casting the gel the DNA was loaded onto the gel after mixing with gel loading buffer and electrophoresed at 5-8V/cm.

Standard precautionary steps were followed while isolating RNA; to
avoid contamination with Rnases.Glass wares used were baked at 200°c
for 5-6 hours. Strict standardization procedures according to the international
norms were followed for the RNA extraction procedure including preparation
of stock solutions, instrument usage and sterilization. The blood sample
was centrifuged and the buffy coat obtained was immediately mixed with
2 ml guanidine isothiocyanate solution. Subsequently, total RNA was extracted
using the acid guanidine isothiocyanate-phenol-chloroform (GITC) method.[15]

RNA electrophoresis was carried on a formaldehyde agarose gel at 6-7
V/cm in the submarine gel electrophoresis system with 1X Formaldehyde
gel running
buffer. RNA was quantified to determine the quality and concentration for
its subsequent use in RT-PCR, by 1ml in alkaline water and reading the
A260 & A280 in a UV spectrophotometer.[16]

In order to remove the contaminating DNA from the RNA, Dnase I treatment
was given, as there should not be any false positive result due to contamination
with the DNA.[17]

The gene specific primers for CK-19 were taken according to Datta et al[9] and
were obtained from Hysel India Limited, New Delhi.

1 mg of Dnase I treated RNA was used for reverse transcription along
with the gene specific primers (CK19 1,CK19 2 and β actin 1 and β actin 2) in a PTC-150 Minicycler (MJ Research). The kit involves the usage of a blend of Moloney variant of Murine Leukemia Virus (M-MLV) with Avian Myeloblastosis virus (AMV) reverse transcriptase on the RNA at gradations of 47°C for 30 minutes, 50°C for 15 minutes and inactivated at 94°C
for 2 minutes and then run at the PCR conditions for 35 cycles with appropriate
positive and negative controls. Subsequently, the samples were loaded on
a DNA gel and viewed in a transilluminator to appreciate any amplification.

The tissues obtained after the incisional biopsies were immediately
processed and stained with haematoxylin and eosin, which confirmed squamous
cell
carcinoma with degrees of differentiation.

RESULTS

Amplification was obtained for the β actin RT-PCR
that was set up for positive control among both the control and study population

However, there was no amplification among the control and study group
for CK19 RT-PCR in both samples obtained before and after incisional
biopsy [Figure - 1], [Figure
- 2], [Figure - 3].

DISCUSSION

Molecular biology has profound influence on the basic understanding
of several disease processes. The study of genetics and its role in causing
human disease is now widely recognised as being at the forefront of medical
research. In itself, this approach has been a departure from conventional
histopathology, where the broad tissue patterns and cytological appearance
observed are a result of the combined expression of tens or thousands
of genes.

There are two types of genes based on their expression patterns:
Housekeeping genes that are expressed in all tissues (eg. β actin)
and tissue specific genes, which have specific functions in specific
tissues (eg. CK19 in oral epithelium). While tissue specific genes are
present in every cell of all tissues, they are expressed by the formation
of mRNA followed by the formation functional protein in specific cells
or tissue. Cytokeratins are one of the main families of intermediate
filaments that are predominantly expressed in epithelial cells where
they show strict lineage and differentiation associated patterns of expression.
Malignant cells generally retain their intermediate filaments of their
progenitor type and consequently cytokeratins have been used to characterize
neoplastic cells. Among these, cytokeratin19, a 19 Kda epithelial cytoskeletal
protein is considered as an epithelial marker as they are not expressed
in normal[17] haematopoietic
tissues. It is believed that malignant epithelial cells are released
into the blood stream during incisional biopsy and these free floating
cells may establish metastasis elsewhere.[18] Our
objective was to test if we can detect these released cells by a sensitive
and powerful method like RT-PCR; an adaptation of PCR. PCR is a sensitive
technique to detect the presence of specific DNA sequences. The preference
of RT-PCR over routine PCR is due to the fact that the closeness of exons
in mRNA reduces the segment to be amplified, especially in situations
where the point of interest in the genome or gene to be amplified is
too large.

Primer design is of paramount importance, as it should specifically
detect the sequence of interest CK19 in this case and not with other
non-specific
entities. Further, they should be specific enough to distinguish between
members of a family of genes or the functional gene and its pseudogene.[19] Our
primers were specific as shown by the amplification of the β actin
and CK19 genes from the isolated genomic DNA and that our PCR technique
is sensitive enough to detect the genes in the human genome. Under the
same optimized conditions, RT-PCR was performed to detect mRNA expression
of CK19 gene. However, no amplification was obtained for the same.

At this juncture, rationale thinking on non-amplification of CK19 gene
expression by RT-PCR has thrown us with many possible considerations for
the same.

Where have the Circulating Cells Gone?

Cancer cells after gaining access to the venous circulation are completely
trapped in the vasculature of the organs of first encounter eg. The lung
and vertebrae for cancers draining into the vena cava. Large numbers of
circulating cancer cells may be killed in the microvasculature by haemodynamic
destruction, by elements of the host surveillance system such as macrophages
and natural killer cells.[20] Nabil
Hanna in her literature review on NK cells has mentioned the discovery
of a fast clearance assay of circulating tumour cells invivo that selectively
detects NK cell mediated tumouricidal activity.[20]

Cytotoxic T Lymphocytes (CTL) recognise antigenic peptides only when they
are presented in association with major histocompatibility ( MHC) class
1 molecules . A lack of MHC class 1 antigens on neoplastic cells would
thus be as effective as a lack of tumour specific antigens in abrogating
or evading immune surveillance.

The cytolytic conjugate between CTLs and tumour cells is stabilized
by interactions between the LFA-1 integrin on the lymphocytes and the
ICAM-1
ligand expressed by the target cell. Lack of expression of ICAM -1, would
appear to permit circulating tumour cells to avoid establishing stable
cytolytic conjugates, and might provide a mean of evading CTL mediated
killing.

Moreover, metastasis is a dynamic and periodic event, and may not be
ongoing at the time of a single blood flow. Interestingly many studies
showed that
the circulating cancer cells were transiently (15 minutes) detectable.[21]

The time interval was followed accordingly in our study. Blood samples
were drawn immediately before biopsy to check if there was any previous
dissemination.

Time intervals between the samples may need to be defined for each
tumour type or in relation to their metastatic potential, as the timing
of samples
in relation to invasive procedures may be critical.[22]

Threshold of RT-PCR Datta et al[9] have discussed
that the lack of CK 19 PCR positivity in the blood of stage IV patients
may have been due to be consistently low levels of expression of circulating
carcinoma cells below the threshold of detection by RT-PCR. However, reports
indicate that the RT-PCR assay can detect a single cancer cell in up to
100 million background cells invitro.[16], [23]

Length of the Expected Fragment Since RT-PCR depends upon the efficiency of reverse transcriptase
as well as DNA polymerase, it is likely that the fragment size was too
large for routine detection. Primers designed close to the 3'end
might have increased the odds of detecting the transcript. But the same
primer design has been used in several studies successfully in detecting
the same CK19 transcript.

Several authors have been successful in detecting occult metastasis
especially in relation to breast carcinoma, lung carcinoma, and prostrate
carcinoma
using the CK 19 RTPCR assay.[8],[9],[10] The
same primer design has been followed in the present study as well .

Integrity of RNA As the conventional method (GITC) method for isolation of RNA was
adopted, the integrity of the RNA may be under question. However, β actin
mRNA amplification authenticated the RNA integrity.

CK19 Expression It is considered that the over expression of CK19 in oral squamous
cell carcinoma generally indicates limited metastatic potential and vice-versa.
Probably the non-amplification of CK19 can be attributed to the fact
that the tumour in our test group patients had over expression of CK19
gene.[24]

Cole et al[26]\ discuss
that the incisional biopsies for surface tumours such as oral cavity,
bronchus
may result in oozing of blood from the surface of the biopsy site.
Since any bleeding from a surface biopsy is not confined, there should
be no
elevation of tissue pressure and therefore no possibility of intravasation
of the cancer cells.

Even if there are viable tumour cells after the incisional biopsy,
the first line of defence like the natural killer cells, nm23 gene
expression
and elaboration of natural inhibitor proteins such as TIMPs plasminogen
inhibitors, will function as metastasis suppressors and prevent tumour
cell invasion. Moreover neoplasms are biologically heterogeneous
and the process of metastasis is selective and depends on a combination
of factors.[27], [28], [29], [30]

Genetic and Environmental Factors Most of the published data is based on the western population
and is related to carcinomas affecting various other organs. There
is only
one published study with regard to oral cancer.[4] Studies
with regard to Indian population with special emphasis on oral cancer
utilizing molecular techniques are very limited. In fact there is
no reported study
on oral cancer and dissemination. As racial, familial and genetic
factors are crucial in cancer progression, they may have played a
vital role
in the non-detection of cancer cells among our study group.

Sample Size The effectiveness of the RT-PCR based assays should
be assessed by considering larger sample size of test
samples.

Newer markers such asCK20 may be more fruitful in reliably detecting
dissemination as few authors suggest that CK19 may not be a very
powerful marker.

However, our patients in the study are so far free of any recurrence
or metastasis after the treatment after being followed up for
the past one
and half years and hence suggesting that the molecular technique
could have been accurate in assessing the dissemination status.

Conclusion

The results of our study show that there is no dissemination of cancer
cells after incisional biopsy into the circulation in oral squamous cell
carcinoma. The patients too are disease free after one and half years of
follow up. However, larger sample size with the use of several markers
would definitely increase the specificity and reliability of these PCR
based assays.